Journal: The EMBO Journal

Article Title: DELE1 maintains muscle proteostasis to promote growth and survival in mitochondrial myopathy

doi: 10.1038/s44318-024-00242-x

Figure Lengend Snippet: ( A ) Model depicts predicted effects of the DELE1 mt-ISR on protein translation including acute inhibition of translation initiation by pS51-eIF2α, resumption of protein initiation following the transcriptional upregulation of Eif3c , and facilitation of translation elongation through increased production of protein synthesis intermediates by the coordinated upregulation of (1) genes related amino biosynthesis and transport, (2) Xpot to mediate tRNA export from the nucleus to the cytosol, and (3) aminoacyl-tRNA synthases (aaRSs) to promote aminoacyl conjugation to tRNAs. Protein translation dynamics and fidelity can affect the proportion of newly translated proteins that fold properly vs. misfold. ( B , C ) Measurement of in vivo protein synthesis in C10 G58R; Dele1 KO mice and indicated littermates at P21 and P28, using the SUnSET assay. Blot for P21 timepoint is shown in ( B ). Mice were injected with the aminoacyl-tRNA ortholog, puromycin, sacrificed 30 min later, and puromycin incorporation into newly synthesized polypeptides in heart and gastrocnemius tissue lysates was measured by immunoblotting. Statistical analysis performed using Welch ANOVA and Dunnett’s T3 multiple comparisons test. (Top graph, heart, Day 21) adjusted p -values were 0.4894 for C10 WT; Dele1+ vs. Dele1 KO, 0.0236 for C10 WT; Dele1+ vs. C10 G58R; Dele1+, 0.5341 for C10 WT; Dele1+ vs. C10 G58R; Dele1 KO, and 0.9978 for C10 G58R; Dele1+ vs. C10 G58R; Dele1 KO. (Top graph, heart, Day 28) adjusted p -values were 0.8419 for C10 WT; Dele1+ vs. Dele1 KO, 0.8923 for C10 WT; Dele1+ vs. C10 G58R; Dele1+, 0.2250 for C10 WT; Dele1+ vs. C10 G58R; Dele1 KO, and 0.4979 for C10 G58R; Dele1+ vs. C10 G58R; Dele1 KO. (Middle graph, gastroc, Day 21) adjusted p -values were 0.9990 for C10 WT; Dele1+ vs. Dele1 KO, 0.1338 for C10 WT; Dele1+ vs. C10 G58R; Dele1+, 0.0136 for C10 WT; Dele1+ vs. C10 G58R; Dele1 KO, and 0.7086 for C10 G58R; Dele1+ vs. C10 G58R; Dele1 KO. (Middle graph, gastroc, Day 28) adjusted p -values were 0.9841 for C10 WT; Dele1+ vs. Dele1 KO, 0.9399 for C10 WT; Dele1+ vs. C10 G58R; Dele1+, 0.7107 for C10 WT; Dele1+ vs. C10 G58R; Dele1 KO, and 0.8419 for C10 G58R; Dele1+ vs. C10 G58R; Dele1 KO. (Top graph, gastroc/heart ratio, Day 21) adjusted p -values were 0.9999 for C10 WT; Dele1+ vs. Dele1 KO, 0.6361 for C10 WT; Dele1+ vs. C10 G58R; Dele1+, 0.2390 for C10 WT; Dele1+ vs. C10 G58R; Dele1 KO, and 0.1941 for C10 G58R; Dele1+ vs. C10 G58R; Dele1 KO. (Top graph, gastroc/heart ratio, Day 28) adjusted p -values were 0.8820 for C10 WT; Dele1+ vs. Dele1 KO, 0.4304 for C10 WT; Dele1+ vs. C10 G58R; Dele1 +, 0.3415 for C10 WT; Dele1+ vs. C10 G58R; Dele1 KO, and 0.1941 for C10 G58R; Dele1+ vs. C10 G58R; Dele1 KO. In all graphs, * indicates p ≤ 0.05 and “ns” not significant. N ≥ 4 mice per group (genotype). Error bars represent SD. ( D ) Representative immunofluorescence images of gastrocnemius muscle from P28 C10 G58R; Dele1 KO mice and indicated littermates, showing fibers with many or confluent aggregates of ubiquitinated proteins co-localized with the aggregate-forming adapter protein p62, suggesting proteostatic collapse (arrowheads) in low power (20×) images (top panels). Scale bars = 50 μm. ( E ) Quantification of ( D ). N = 3 mice per genotype with 30 low power fields counted per genotype. Low-power field size was 397.75 μm × 397.75 μm. Statistical analysis performed using ordinary one-way ANOVA with Šidák’s multiple comparisons test, with a single pooled variance. N = 3 in each group. P = 0.8857 for C10 WT; Dele1+ vs. C10 WT; Dele1 KO. P = 0.6605 for C10 WT; Dele1+ vs. C10 G58R; Dele1 +. ns = non-significant. **** =< 0.0001. Error bars represent SD. ( F ) Representative immunofluorescence images of gastrocnemius muscle from P28 C10 G58R; Dele1 KO mice injected with puromycin 30 min prior to sacrifice as in ( B ). Muscle cross-sections were immunostained for ubiquitinated proteins (using the FK2 antibody) (green), puromycin (red), and laminin (blue). Arrowheads indicate muscle fibers containing many or confluent aggregates of ubiquitinated proteins that were also co-stained for elevated puromycylated polypeptides. Arrows indicate individual aggregates of ubiquitin proteins that also contain puromycylated polypeptides. Scale bars = 20 μm. ( G , H ) Quantification of ( F ). Individual myofibers were automatically segmented using the Laminin immunofluorescence to define the myofiber border. Average puromycin fluorescence intensity and cross-sectional area (CSA) were measured for each myofiber. Separately myofibers were manually scored as containing many or confluent aggregates of ubiquitinated proteins (Ub+ or Ub- muscle fibers, respectively). The average puromycin intensity for Ub+ and Ub- myofiber is shown in graph separately for three mice (m1–3). N = 3 mice with 10 high power fields counted per mouse. The scatterplot in ( H ) shows the relationship between myofiber CSA and puromycin intensity for all myofibers analyzed in ( G ). Fibers with CSA < 50 μm were excluded from analysis. Statistical analysis was performed using Kruskal–Wallis test with Dunn’s multiple comparisons test as data was non-parametrically distributed for ( G ). ( I ) Representative confocal images show three consecutive sections through two areas of gastrocnemius from C10 G58R; Dele1 KO mice triple stained for muscle fiber type 1, 2a, or 2b (red) and FK2 to detect ubiquitin protein aggregates (green) and a sarcolemma marker protein laminin or dystrophin (blue). Fiber type 2x is defined by the absence of any other fiber marker in the consecutive sections. Areas 1 and 2 are magnifications of the boxed areas in Appendix Fig. , which shows the whole tissue section. Scale bar = 100 μm. Yellow arrow = type 1 fiber, solid white arrowhead = type 2a fiber, open white arrowhead = 2b fiber, and solid white arrow = 2x fiber. ( J ) Graph quantifying the CSA of fibers with and without aggregates of ubiquitinated proteins, separated by fiber type as in ( I ). Gastrocnemius from three C10 G58R; Dele1 KO mice were analyzed. Statistical analysis was performed using Kruskal–Wallis test with Dunn’s multiple comparisons test as data was non-parametrically distributed. ( K ) Graph quantifying the proportion of each fiber types that contained aggregates of ubiquitinated proteins from tissue stained as in ( I ). Gastrocnemius from three C10 G58R; Dele1 KO mice were analyzed. Statistical analysis was performed using Kruskal–Wallis test with Dunn’s multiple comparisons test as data was non-parametrically distributed. N = 3 in each group. P = 0.4081 for percent of type 1 myofibers with FK2 aggregates vs. percent of type 2a myofibers with FK2 aggregates. P = 0.0338 for percent of type 1 myofibers with FK2 aggregates vs. percent of type 2b myofibers with FK2 aggregates. P = 0.8902 for percent of type 1 myofibers with FK2 aggregates vs. percent of type 2b myofibers with FK2 aggregates. ns = non-significant. * = <0.05. Error bars represent SD. .

Article Snippet: Rabbit monoclonal anti-Ubiquitin FK2 , StressMarq , SMC-550D.

Techniques: Inhibition, Conjugation Assay, In Vivo, Injection, Synthesized, Western Blot, Immunofluorescence, Staining, Fluorescence, Marker

Journal: The EMBO Journal

Article Title: DELE1 maintains muscle proteostasis to promote growth and survival in mitochondrial myopathy

doi: 10.1038/s44318-024-00242-x

Figure Lengend Snippet: ( A ) Representative immunofluorescence images of gastrocnemius muscle from C10 G58R; Dele1 KO and indicated littermates triple-stained for the ubiquitinated protein marker, FK2 (green), the aggregate adapter protein p62 (red) and laminin (blue). In the C10 G58R; Dele1 KO genotype, a subset of fibers displayed FK2 and p62 colocalized in individual (white arrows) or confluent aggregates (arrowheads), suggesting proteostatic collapse. High power (60X) views of the boxed areas are shown in bottom panels. Scale bars = 10 μm. Note: animals were not injected with puromycin in this experiment. ( B ) Quantification of ( A ). Aggregates positive for both FK2 and p62 were counted in 10 high power (60X) fields. N = 3 mice per genotype with 29 or 30 fields counted total per sample. High-power (60X) field size is 132.58 μm × 132.58 μm. Statistical analysis was performed using the Kruskal–Wallis test with Dunn’s multiple comparisons test, as the data distribution was non-parametric. **** indicates p ≤ 0.0001 and “ns” not significant. Adjusted p -values are >0.9999 for WT vs. Dele1 KO, >0.9999 for WT vs. C10 G58R, <0.0001 for WT vs. C10 G58R; Dele1 KO, and <0.0001 for C10 G58R vs. C10 G58R; Dele1 KO. ( C ) Representative immunofluorescence images of gastrocnemius muscle from P28 C10 G58R; Dele1 KO mice and indicated littermates injected with puromycin 30 min prior to sacrifice as in (Fig. ). Muscle cross-sections were immunostained for the ubiquitinated protein marker, FK2 (green), puromycin (red), and laminin (blue). Arrowheads indicate muscle fibers containing many or confluent aggregates of ubiquitinated protein that were also co-stained for elevated puromycylated polypeptides. N = 1 mouse for each genotype except for P28 C10 G58R; Dele1 KO mice for which N = 3 mice. Scale bars = 20 μm. ( D ) Quantification of myofiber cross-sectional area (CSA) as in (Fig. ). The average CSA for Ub+ and Ub- myofiber is shown in graph separately for three G58R; Dele1 KO mice (m1–3) in graph. N = 3 mice with 10 high power fields counted per mouse. Statistical analysis was performed using the Kruskal–Wallis test with Dunn’s multiple comparisons test, as the data distribution was non-parametric. ** and **** indicates p ≤ 0.01 and 0.0001, respectively, and “ns” not significant. P -values (from left to right) are 0.0055, <0.0001, and 0.0016. ( E ) Schematic summarizing data showing that Dele1 KO results in variable protein synthesis, decreased proteostasis, and increased muscle fiber atrophy within skeletal muscle undergoing mitochondrial stress. We hypothesize that these changes are responsible for the decreased growth and survival in MM models with early mitochondrial stress, in the absence of the Dele1 mt-ISR.

Article Snippet: Rabbit monoclonal anti-Ubiquitin FK2 , StressMarq , SMC-550D.

Techniques: Immunofluorescence, Staining, Marker, Injection

Journal: The EMBO Journal

Article Title: DELE1 maintains muscle proteostasis to promote growth and survival in mitochondrial myopathy

doi: 10.1038/s44318-024-00242-x

Figure Lengend Snippet: Reagents and tools table

Article Snippet: Rabbit monoclonal anti-Ubiquitin FK2 , StressMarq , SMC-550D.

Techniques: Recombinant, Control, Sequencing, Electron Microscopy, Saline, Staining, Microarray, Software, Laser-Scanning Microscopy, High Performance Liquid Chromatography, Mass Spectrometry

Journal: bioRxiv

Article Title: The Shigella flexneri effector IpaH1.4 facilitates RNF213 degradation and protects cytosolic bacteria against interferon-induced ubiquitylation

doi: 10.1101/2024.09.05.611450

Figure Lengend Snippet: (A - C) Cells were primed with 100 U/ml IFNγ or left unprimed and then infected with WT or ΔmxiE S. flexneri at an MOI of 50 - 100. Cells were fixed 3 - 4 h post infection (hpi) and stained for linear ubiquitin (M1-Ub) with anti-M1 antibody. Percentage of M1-Ub-positive bacteria was quantified in infected A549 epithelial cells at 4 hpi (B) and HT29 epithelial cells at 3 hpi (C) . Graphs show the average of three independent experiments and depict means ± SEM. Two-way ANOVA with Tukey’s multiple comparison tests were performed; all statistically significant comparisons are shown. ***p<0.001; **** p<0.0001; ns, not significant.

Article Snippet: The following primary antibodies were used: rabbit monoclonal anti-RNF213 (Sigma HPA026790; 1:1000), rabbit monoclonal anti-linear (M1) ubiquitin; clone 1E3 (Sigma ZRB2114; 1:250), mouse polyclonal anti-ubiquitin FK2 (Cayman Chemical 14220; 1:100), rabbit monoclonal anti-K63 ubiquitin, clone Apu3 (Sigma 05-1308.

Techniques: Infection, Staining, Bacteria, Comparison

Journal: bioRxiv

Article Title: The Shigella flexneri effector IpaH1.4 facilitates RNF213 degradation and protects cytosolic bacteria against interferon-induced ubiquitylation

doi: 10.1101/2024.09.05.611450

Figure Lengend Snippet: (A) WT A549 cells expressing the indicated internally Strep-tagged Ubiquitin (INT-Ub) variants were primed with 100 U/ml IFNγ overnight and co-localization of INT-Ub with cytosolic S. flexneri (Sf) Δ mxiE was assessed at 4 hpi. Ubiquitin linkage-specific antibodies were used to determine the percentage of S. flexneri Δ mxiE staining positive for K27-linked ubiquitin (K27-Ub) (B) or K63-linked ubiquitin (C) in IFNγ-primed untransduced WT A549 cells. Co-staining of anti-M1 and anti-ubiquitin (FK2) on the surface of S. flexneri Δ mxiE in IFNγ-primed (100U/ml) A549 cells is shown in (D). Quantification of anti-M1-FK2 co-staining in untreated and IFNγ-primed A549 cells is depicted in (E). All infections were done at an MOI of 50 - 100. Data was generated from at least three independent experiments and shows mean ± SEM. One-way ANOVA followed by Dunnet’s multiple comparisons were performed of all groups against WT-ubiquitin group (A) . Two-way ANOVA with Tukey’s multiple comparison tests were performed for (B) and (C) . An unpaired t-test was performed between “both FK2+M1” groups (gray bars) ( E). *p<0.05; **p<0.01; ***p<0.001; **** p<0.0001

Article Snippet: The following primary antibodies were used: rabbit monoclonal anti-RNF213 (Sigma HPA026790; 1:1000), rabbit monoclonal anti-linear (M1) ubiquitin; clone 1E3 (Sigma ZRB2114; 1:250), mouse polyclonal anti-ubiquitin FK2 (Cayman Chemical 14220; 1:100), rabbit monoclonal anti-K63 ubiquitin, clone Apu3 (Sigma 05-1308.

Techniques: Expressing, Staining, Generated, Comparison

Journal: bioRxiv

Article Title: The Shigella flexneri effector IpaH1.4 facilitates RNF213 degradation and protects cytosolic bacteria against interferon-induced ubiquitylation

doi: 10.1101/2024.09.05.611450

Figure Lengend Snippet: (A) Immunoblotting for HOIP, HOIL-1, and RNF213 protein expression in untreated and IFNγ-primed WT and the corresponding gene deletion (KO) A549 cells. (B) Percentage of M1-linked ubiquitin positive Δ mxiE S.flexneri in IFNγ-primed WT, HOIP KO , HOIL-1 KO and RNF213 KO A549 cells. (C) Percentage of WT and 7KR INT-Ub-positive Δ mxiE S. flexneri in IFNγ-primed WT, HOIP KO , and RNF213 KO A549 cells. (D – F) Untreated and IFNγ-primed A549 and HT29 cells were infected with the indicated S. flexneri strains and immuno-stained for RNF213 and ubiquitin (FK2). Representative immunofluorescence microscopy images are shown for A549 infections in (E). RNF213- S. flexneri colocalization percentages were quantified in A549 (D) and HT29 (F) cells. (G) Quantification of ubiquitin and RNF213 colocalization with Δ mxiE S. flexneri in A549 cells. Percentages of Δ mxiE S. flexneri staining positive for antibodies specific for K27-linked (H) and K63-linked ubiquitin (I) are also provided. All data are represented by the mean ± SEM from at least three independent experiments. One-way ANOVA followed by Dunnet’s multiple comparisons were performed of all groups against WT A549 group in (A) . Two-way ANOVA with Tukey’s multiple comparison tests were performed in (C, D and F) . an unpaired t-test was performed between “both Ub+RNF213” groups (gray bars) in (G) . For (H and I) , an unpaired-t test was performed. **p<0.01;; **** p<0.0001.

Article Snippet: The following primary antibodies were used: rabbit monoclonal anti-RNF213 (Sigma HPA026790; 1:1000), rabbit monoclonal anti-linear (M1) ubiquitin; clone 1E3 (Sigma ZRB2114; 1:250), mouse polyclonal anti-ubiquitin FK2 (Cayman Chemical 14220; 1:100), rabbit monoclonal anti-K63 ubiquitin, clone Apu3 (Sigma 05-1308.

Techniques: Western Blot, Expressing, Infection, Staining, Immunofluorescence, Microscopy, Comparison

Journal: bioRxiv

Article Title: The Shigella flexneri effector IpaH1.4 facilitates RNF213 degradation and protects cytosolic bacteria against interferon-induced ubiquitylation

doi: 10.1101/2024.09.05.611450

Figure Lengend Snippet: (A) IFNγ-primed A549 cells were infected with the indicated PilT + S. flexneri strains for 3 hours at an MOI of 5 - 25 and RNF213 protein levels were assessed by Western blotting. (B) Representative microscopy image depicting RNF213 recruitment to cytosolic Δ IpaH1.4 bacteria in IFNγ-primed A549 cells. (C) Percentage of RNF213 + WT and Δ IpaH1.4 S. flexneri in IFNγ-primed A549 cells. (D) Representative microscopy image showing Δ IpaH1.4 S. flexneri decorated with M1-linked ubiquitin in IFNγ-primed A549 cells. Percentage of M1-linked (E) , K27-linked (F) , and K63-linked (G) ubiquitin-positive WT and Δ IpaH1.4 S. flexneri in IFNγ-primed WT (E-G) and RNF213 KO (E) A549 cells. (H) The ilux operon was introduced into S. flexneri strains to use bioluminescence (RLU) as a proxy for bacterial growth. RLU was measured in IFNγ-primed WT A549 cells infected with the indicated S. flexneri strains at an MOI of 5. (C, F-H) Data represent the mean ± SEM from at least three independent experiments. An unpaired t-test was conducted for (C, F, G) . One-way ANOVA followed by Dunnet’s multiple comparisons were performed between all groups against the group of WT A549 cells (E). *p<0.05; ***p<0.001; **** p<0.0001.

Article Snippet: The following primary antibodies were used: rabbit monoclonal anti-RNF213 (Sigma HPA026790; 1:1000), rabbit monoclonal anti-linear (M1) ubiquitin; clone 1E3 (Sigma ZRB2114; 1:250), mouse polyclonal anti-ubiquitin FK2 (Cayman Chemical 14220; 1:100), rabbit monoclonal anti-K63 ubiquitin, clone Apu3 (Sigma 05-1308.

Techniques: Infection, Western Blot, Microscopy, Bacteria

Journal: BMC Biology

Article Title: Identification of a novel DNA oxidative damage repair pathway, requiring the ubiquitination of the histone variant macroH2A1.1

doi: 10.1186/s12915-024-01987-x

Figure Lengend Snippet: mH2A is ubiquitinated upon oxidative stress. A Stable HeLa cell lines expressing e-mH2A1.1 were treated with H 2 O 2 and allowed recovering for the times indicated. The e-mH2A1.1 nucleosomal complex was immunopurified, run on a gel containing SDS and proteins were identified by mass spectroscopy. The positions of PARP-1, non-modified and mono(ub1)- and bi-ubiquitinated(ub2) e-mH2A1.1, histone H1 and core histones are indicated. B Western blot of the e-mH2A.1.1 complex. The blot was first revealed with anti-PARP-1 antibody and then with anti-HA antibody for visualization of mH2A1.1. C Aminoacid sequence of mH2A1.1 encompassing AA 109–132. The positions of the two identified by mass spectrometry ubiquitination sites (I) and (II), corresponding to lysine residues 115 and 120, are indicated. D Western blot analysis of the e-mH2A.1.1 complex immunoprecipitated either with anti-IgG or with anti-ubiquitin FK2, and blotted with an anti-FLAG antibody

Article Snippet: Antibodies employed were as follows: monoclonal anti-Flag antibody M2 (Sigma), monoclonal anti-HA antibody 9E (Roche), monoclonal anti-PARP-1 ALX-804-211 (Enzo Life Sciences), monoclonal anti-ADP-ribose ALX-804-220 (Enzo Life Sciences), polyclonal anti-mH2A1 (Stefan Dimitrov, INSERM, Grenoble, France), polyclonal anti-mH2A1 ab37264 (Abcam), and polyclonal anti-H2A ab18975 (Abcam), anti-Ubiquitin mouse mAb (FK2) ST1200 (Sigma), Anti-Mouse IgG A6531 (Sigma).

Techniques: Expressing, Mass Spectrometry, Modification, Western Blot, Sequencing, Immunoprecipitation

Journal: Neuron

Article Title: Rapid iPSC inclusionopathy models shed light on formation, consequence, and molecular subtype of α-synuclein inclusions

doi: 10.1016/j.neuron.2024.06.002

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: Anti-Ubiquitin (FK2) , EMD Millipore , Cat. # ST1200; RRID:AB_10681625.

Techniques: Transduction, CRISPR, Sequencing, Software